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Initiation, extension, and termination of RNA synthesis by a paramyxovirus polymerase
- Source :
- PLoS Pathogens, Vol 14, Iss 2, p e1006889 (2018), PLoS Pathogens
- Publication Year :
- 2018
- Publisher :
- Public Library of Science (PLoS), 2018.
-
Abstract
- Paramyxoviruses represent a family of RNA viruses causing significant human diseases. These include measles virus, the most infectious virus ever reported, in addition to parainfluenza virus, and other emerging viruses. Paramyxoviruses likely share common replication machinery but their mechanisms of RNA biosynthesis activities and details of their complex polymerase structures are unknown. Mechanistic and functional details of a paramyxovirus polymerase would have sweeping implications for understanding RNA virus replication and for the development of new antiviral medicines. To study paramyxovirus polymerase structure and function, we expressed an active recombinant Nipah virus (NiV) polymerase complex assembled from the multifunctional NiV L protein bound to its phosphoprotein cofactor. NiV is an emerging highly pathogenic virus that causes severe encephalitis and has been declared a global public health concern due to its high mortality rate. Using negative-stain electron microscopy, we demonstrated NiV polymerase forms ring-like particles resembling related RNA polymerases. We identified conserved sequence elements driving recognition of the 3′-terminal genomic promoter by NiV polymerase, and leading to initiation of RNA synthesis, primer extension, and transition to elongation mode. Polyadenylation resulting from NiV polymerase stuttering provides a mechanistic basis for transcription termination. It also suggests a divergent adaptation in promoter recognition between pneumo- and paramyxoviruses. The lack of available antiviral therapy for NiV prompted us to identify the triphosphate forms of R1479 and GS-5734, two clinically relevant nucleotide analogs, as substrates and inhibitors of NiV polymerase activity by delayed chain termination. Overall, these findings provide low-resolution structural details and the mechanism of an RNA polymerase from a previously uncharacterized virus family. This work illustrates important functional differences yet remarkable similarities between the polymerases of nonsegmented negative-strand RNA viruses.<br />Author summary RNA viruses replicate and transcribe their genomes using complex enzymatic machines known as RNA-dependent RNA polymerases. The chemical reactions driving nucleotide addition are shared among nucleic acid polymerases but the underlying mechanisms of RNA biosynthesis and the complex polymerase structures are diverse. Of these RNA viruses is the paramyxovirus family, which includes major human pathogens. Paramyxoviruses have common biological and genetic properties but little is known about their replication machinery. Insights into the structure, function, and mechanisms of RNA synthesis of one paramyxovirus polymerase will likely extend to the entire virus family. An emerging, highly pathogenic paramyxovirus is Nipah virus (NiV), which causes encephalitis in humans. We have purified NiV polymerase, probed its enzymatic and biophysical properties and developed it as a model paramyxovirus polymerase. We investigated template strand sequence elements driving RNA biosynthesis for NiV polymerase and obtained a snapshot of NiV polymerase molecular organization using electron microscopy to provide the first structural information on a paramyxovirus polymerase. This work extends previous knowledge by producing the first recombinant paramyxovirus polymerase and using this protein in enzymatic assays to highlight key functional and structural characteristics for the design of new medicines.
- Subjects :
- RNA viruses
0301 basic medicine
Transcription Elongation, Genetic
Polyadenylation
viruses
Gene Expression
Pathology and Laboratory Medicine
Virus Replication
Biochemistry
Polymerases
Database and Informatics Methods
chemistry.chemical_compound
RNA polymerase
Medicine and Health Sciences
Macromolecular Structure Analysis
lcsh:QH301-705.5
Transcription Initiation, Genetic
Polymerase
biology
DNA-Directed RNA Polymerases
Nucleic acids
Vesicular Stomatitis Virus
Medical Microbiology
Viral Pathogens
Viruses
Paramyxovirinae
RNA, Viral
Pathogens
Sequence Analysis
Research Article
lcsh:Immunologic diseases. Allergy
Protein Structure
Bioinformatics
Nucleic acid synthesis
Immunology
Polymerase stuttering
Microbiology
Rhabdoviruses
Virus
Viral Proteins
03 medical and health sciences
Sequence Motif Analysis
Virology
DNA-binding proteins
Genetics
Chemical synthesis
Amino Acid Sequence
RNA synthesis
Microbial Pathogens
Molecular Biology
Biology and life sciences
Organisms
Nipah Virus
Proteins
RNA
RNA virus
Phosphoproteins
biology.organism_classification
Research and analysis methods
Biosynthetic techniques
030104 developmental biology
lcsh:Biology (General)
chemistry
Viral replication
Transcription Termination, Genetic
Paramyxoviruses
biology.protein
Parasitology
lcsh:RC581-607
Subjects
Details
- ISSN :
- 15537374
- Volume :
- 14
- Database :
- OpenAIRE
- Journal :
- PLOS Pathogens
- Accession number :
- edsair.doi.dedup.....ad819dae6dcc6d9a2cded96bf550c6ac
- Full Text :
- https://doi.org/10.1371/journal.ppat.1006889